Topical administration of vitamin b-12

ABSTRACT

A VITAMIN B-12 IS ADMINISTERED TO ANIMALS BY TOPICAL APPLICATION IN A NON-TOXIC SOLVENT.

United States Patent 3,577,537 TOPICAL ADMINISTRATION OF VITAMIN B-12 Eugene E. Howe, Somerset, and Charles Rosenblum, Princeton, N.J., assiguors to Merck & Co., Inc., Rahway,

3,577,537 Patented May 4, 1971 "ice that might be mentioned are organic solvents such as alcohols, for example, ethanol, water, and mixtures of these solvents. The concentration of the Vitamin B-12 in the solvent is not critical and can be varied depending upon the particular solvent. In general, it is found that NJ. 5 N0 Drawing continuationdmpart of application S91. No. solutions containing from 0.5 pg. to 1,000 g. of Vitamin 629,918, Apr. 11, 1967. This application Mar. 9, 1970, p are satlsfactory, gq concen- Ser. No. 17,897 trations up to 10,000 ig. can be utilized if desired.

Int. Cl. A61k 25/02 In carrying out the method of this invention, the solu- 424-201 4 Clalms tion of Vitamin B12 is applied to the skin, whereupon the Vitamin B-12 is absorbed by the skin. The amount absorbed will vary somewhat, depending on the individual ABSTRACT OF THE DISCLOSURE animal and the area upon which the solution of the Vitamin B-12 is administered to animals by topical vitamin is applied. The vitamin absorbed by the skin application inanon-toxic solvent. 15 is slowly released to other tissues of the body, the skin thus acting as a depot or reservoir for the Vitamin B-12. The following experiments carried out in rats and BACKGROUND OF THE INVENTION guinea pigs demonstrate the absorption of Vitamin B-12 by topical applications of solutions of the vitamins. Y 9 1s cancel-neg wlth a m of.adIlnn Experiments were performed to determine whether staring Y p B 12 to ammals' Thls apphcatlon 1S a cyanocobalamin applied to the skin in water would stimui i gg ggg gg g iig filed late growth of weanling rats consuming a Vitamin B-12 B 12 (1m m b b d f the deficient diet. In these experiments, male rats of the a i tr Y .8 l t the HoltZn-ian strain were separated into groups of 8, housed Ess x 2537 2 :5; 1; a E s g S 3 ineclr 25 individually and allowed free access to food and water. 1 i p The diet consisted of 60% soy bean meal, 23.9% glucose, anism to tacili tate its absorption. It has been reported 4% Salt mixture 10% partially hydrogenated fat, 2% i the vltamm absorbed Subhnguany nasal cod liver oil 0.1% thyroid powder and a vitamin addeninhalation, but neither of these methods is being used dum lacking; only Vitamin Such a diet it had 95 3}; i g ggg g 2223 223 g gzfigg g gi previously been established that 0.1 ,ug. cyanocobalamin irlltramuscular injection p y y per rat per day usually produced a maximum growth response. On the assumption that only a small fraction SUMMARY OF THE INVENTION of the topically applied vitamin would be absorbed, 5 g. f cyanocobalamin in 0.03 ml. of water was applied daily It is one ObjfiCt of this invention to provide an improved 9 2 method of administering Vitamin B-12 to animals which Eg i g g s g x 5 i 2; will avoid the difficulties and disadvantages of administerf ii f s i g y Es y i sacrificed ing it parenterally. to a of assassins;arrests its a: Vitamin B-12 to anima s where y t e vitamin is a 40 I Sorbed and Slowly released the rat, the experiments being identical in all other re In accordance with this invention, it is found that Vita- Spects' TABLE 1 min B 12 can be adm,ln1,stered to aqlmals by app,lymg ExperimentI.-Efiect ofsug.cyanocobalamlnappliedtoskinongrowth a solution of the vitamin in a non-toxic solvent topically of weaning rats to the skin of the animal. The skin is found to act as a Average,28 reservoir, absorbing large amounts of the vitamin and day we ip P 1 releasing it slowly to other tissues. D16 gami ue Basal 15651141 DESCRIPTION OF THE PREFERRED Basal plus 0.1 thyroil powieni 134;l=23.8

B 1 0. th 10 EMBODIMENTS iiiaiaiin/ kglht,.ifllfffffi.it???" 182i36.2 0.01 The vitamin can apglied topically to i 8km iitaiiiiiiriilifl. iitiiflgiiiieii'ifibiiii. issiim 0.01 in the form of a solution in suitable solvents which are non-toxic to the animal. Solvents suitable for this purpose 1 Elght mg per gmull TABLE 2 Etlect of 0.1 g. cyanocobalamin applied to skin on growth of weaning rats Average 1 28-day weight gai11=l;S.D. P-value Experiment V. II III II III l asal plus 0.1 thyroid powder 139;|:19.4 1l6;l;22. Basal plus 0.1 thyroid powder plus 0.1 g. cyanocobalam n in 0.p3 m1. HOH applied topically 16019. 9 142:1:22. l 0. 05 Basal plus 0.1 thyroid powder Plus 0.1 pg. cyanocobalamin administered subcutaneously in aqueous solution i15. 5 162121. 2 0.05 O. 01 Basal plus 0.1 thyroid powder Plus 10 g. cyanocobalamin/kg. of diet 1525131. 8 1645:12. 8 0. 01

1 Eight rats per group.

The average 28-day weight gains of the growth Experiments I-III are shown in Tables 1 and2' together with standard deviation (S.D.). Also reported are probability (P) values of differences, based on comparisons with basal diet+ 0.1 thyroid powder, calculated by the method of analysis of variance. Growth responses obtained (Table 2) with daily topical application of 5 ,ug. of cyanocobalamin were significant (P=0.01) and equivalent to ad libitum feeding of gJkg. diet. Even the low dose of 0.1 ,ug. per day when applied topically to the skin caused a maximum growth response.

In order to quantitate the effect more accurately, experiments were performed using Vitamin B-12 Co. In Experiment IV absorption from water and ethanol was Group average absorption results as Percent of Dose of Experiment IV are recorded in Table 3. These data show absorption from the 2 vehicles but that absorption from ethanol is definitely superior to that from water. The apparent lower total absorption observed at 5 days as compared to 2 days is probably due in most part to variations between animals as witness the high incidence of large S.D. values. Such variability is not encountered frequently in later experiments. This may also be due to variable retention of vitamin by the bandage.

In rat Experiment V absorption of cyanocobalamin from ethanolic solution was compared at 4 concentrations (0.025, 0.25, 2.5 and ,ug./0.03 ml.) at 3 contact times: 2 hours, 2 days, and 5 days.

TABLE 4 Experiment V.Efiect of concentration and time on percutaneous absorption by the rat of cyanocobalamin applied in 00 ethanolic solution Average 1 percent of doseiSD. Total absorption Percent of Equivalent, Concentration, 1g. Duration Skin Urine Feces Carcass dose pg.

0.025 2hours 14. 810.3 1. 1511. 53 15. 915. 78 0. 004010. 0015 d 3. 8. 412. 28 0. 02110. 000 8. 211. 0 8. 411. 54 0. 2110. 04 0112.2 1. 2011. 20 10311.87 2.01047 3days 19.41100 01410.07 07710.51 24911.17 22.8111. 28 00057100028 3. 511.7 0 1210.07 01010.11 0. 4310. 20 4. 211. 78 0.0110. 004 7. 712.5 0 2210. 24 0. 5810. 54 1. 4310.91 9. 913. 50 0. 2510. 09 5 012.0 0 101011 1.0410. 30 28811.00 0113.85 2.51007 27 515.3 0 3110. 53 1. 8312. 00 2. 5713. 57 32. 2112. 00 00080100030 9. 915.3 1.4011. 50 00214.04 4. 5111. 48 20. 410. 01 0. 05010. 018 13 015.1 0.821072 2. 8412. 34 5.11100 21. 7110. 0. 5410. 20 .5:|:7.8 03110.17 04913.45 3. 4011. 37 20. 8110. 31 0. 712. 58

1 Four rats per group.

measured using two concentrations, 0.025 3g. and 2.5 ,ug. 40 The data obtained in the performance of Experiment cyanocobalamin in 0.03 ml. of solution, and two Contact V are compiled in Table 4 and show that the dose-response periods, 2 and 5 days. The SO II O tS, h eontamlhg (total absorption) curves are approximately of a log-log several tenths of a m C O 0f ladloaetlvlty P dose, type, characteristic of an absorption process. This is true were PP to 21 Shaved g on the 3 of F 3 of all 3 time intervals studied and the slopes of the log- Holtzman rats i i mg g 5 2 3? i g log curves are about equal. Initial absorption is rapid and were W y gau e g P y remains essentially constant between 2 hours and 2 days, hesrve tape clrclmg the bod1es of the ammals. The rats b tin t 1 2 5 f M b t 2 d 5 d were housed individually in metabolism cages to perm1t s i m l e f b1 e an th the collection of urine and feces, and were allowed unreua Sorphoh 1S qulte valla llt u d r e 0011- stricted access to laboratory chow and water. After the dftlons of the exlfenmeht e h pe to he a funcdesignated time, they were sacrificed, the skin was excised hon of the q y 0f the Vltamln Ep ffem the area of pp f the bandage and Experiment VI was designed to investigate the role of section were washed with portions of detergent solution. the Skin as a depot or mservoir f Vitamin 1 ft r l 2 and bandage w were i l g :3 topical application. Four groups of 5 rats each were treated 100 m Wlt Water as W urme Samp an e c as previously described with 0.03 ml. of ethanolic solusamples were suspended 1n 100 ml. water for assay. A11 tion In 2 5 of m (1.0a ti e c anocobalamin radiometric measurements were made by gamma-ray a 1 g l C v y scintillation counting. Urine, feces, washings and whole The ah1ma1S Were sacrlficed at 19 days and carcasses were measured in a Tobor large volume counter, the appropriate measurements made. Urlne and fe e of while the washed skin samples were measured in the all surviving animals were collected daily. The results of Autogamma instrument for small volume.

TABLE 3 this experiment are shown in Table 5.

Experiment IV.Efiect of solvent on pertlrlltanajous absorption of cyanocobalamin 00 in e ra \ Six rats per group.

TABLE 5 Experiment VL-Percutaneous absorption of cyanocobalamln Co (2.5 g.) showing depot efiect Duration of experiment, days Average 1 percent of dose=l=S.D. 5 40 Urine and feces:

Day 1-5 0.09=|=0.03 0.08:|;0.04 0.06i0.05 54 0. 281:0. 37

1 Five rats per group.

Table 5 is a compilation of group average values obtained in Experiment VI again expressed as Percent of to show that the observed absorptive process is not peculiar to the rat. Ten female guinea pigs of approximately 300 gm. in weight were subjected to experimental procedures similar in all details to those of Experiment VI with the following exceptions. One animal died on the sixth day and was examined at the time of death. 4 were sacrified on the 13th day, and the remaining 5 were sacrificed on the 24th day.

The results of Experiment VII are summarized in Table 6. Despite great variability between animals, the carcass and excreta activities of the guinea pig rise at the expense of a diminishing skin radioactivity, just as was observed with the rat. This behavior indicates that the skin of the guinea pig also serves as a depot source for Vitamin B-l2. Values for skin plus bandage washings are recorded in Tables 5 and 6 as an indication of the radioactivity accountability achieved in these experiments. Although somewhat variable, an average of 96% of the activity is accounted for.

TABLE 6 Experiment VIL-Percutaneous absorptlon oi cyanoeobalamln Co (2.5 pg.) from ethanol solution by the guinea pig as percent of dose Percent of dose Total Wash plus Guinea pig number Skin Carcass U plus F retention 1 bandage Total Average;i;S D 10. 6&621 1.03i0. 97 0.66i0.94 12.3=l=5. 17 89 514. 05 101.8=!;6 53

A erageiS,D 2. 5711.80 1.88iL78 2. 78=l-.4. 10 7. 2:0:7. 45 92.1519. 97 99.3:e4.14

1 Sum of skin, carcass and U plus F values.

1 Died on 6th day.

3 Numbers 1-4 sacrificed on 13th day. 4 Numbers 5-9 sacrificed on 24th day.

Dose. It is clear from these figures that the skin acts as a storage depot for Vitamin B-12, gradually releasing the vitamin for use in other body tissues. This is shown by: (l) the gradual decrease in skin radioactivity with time, by (2) the concomitant rise in carcass activity, and by (3) the continuous elimination of radioactivity in urine plus feces. Thus, increases in elimination of radioactivity in excreta and increases in carcass concentrations occur at the expense of a parallel decrease in skin radioactivity. Excretion during the first 5 days is negligible, but increases thereafter to reach a maximum in about 20 days and is still guite high days after topical application. Animals vary considerably in the permeability and retentivity of their skin to radioactive Vitamin B-12. In many cases, hgh skin retentivity is accompanied by low urinary output.

The purpose of the seventh and final experiment was What is claimed is:

1. A method of administering a therapeutic amount of Vitamin B-l2 to animals which comprises applying a solution of Vitamin B-12 in a non-toxic liquid solvent to the skin of said animal.

2. The method of claim 1 wherein the solvent is Water.

3. The method of claim 1 wherein the solvent is a nontoxic alcohol.

4. The method of claim 1 wherein the solvent is ethanol.

References Cited Schaeler et al. JL. Nutr. 59(2):17l179, June 1956, Absorption of Topically Applied Vitamins.

Okuda et al. Proc. Soc. Exp. Biol. Med. 123 (2):504- 506, November 1966, Absorption of Vitamin B in a Rectal Suppositor.

SHEP K. ROSE, Primary Examiner 

